Hypertension is a multi-factorial, pathogenic process associated with a number of occlusive vascular diseases including myocardial infarction, stroke, and end-stage renal failure (Lifton, R. P. (1995) Proc. Nat. Acad. Sci. 92:8545-51). Essential (or primary) human hypertension, as opposed to the more rare monogenetic forms, appears to be controlled by genetic and epigenetic events. To date, several forms of monogenetic (Mendelian) human hypertension have been reported, where single gene defects result in a hypertensive phenotype in the vast majority of affected individuals. These include pseudoaldosteronism (Liddle syndrome, described in Shimkets, R. A. et al. (1994) Cell 79:407-14), glucocorticoid-remediable aldosteronism (GRA, described in Lifton, et al. (1992) Nature. 355:262-5), and most recently apparent mineralocorticoid excess (AME, described in Mune et al. (1995) Nat. Gen. 10(4):394-9), and pseudohypoaldosteronism type II (Gordon syndrome, described in Gordon et al. (1995) Raven, N.Y., pp. 2111-23).
Evidence which supports the influence of heredity in essential hypertension includes epidemiologic studies, which demonstrate significant familial aggregation of blood pressure (Longini, et al. (1984) Am. J. Epidemiol. 120:131-44.) This is attributable to a genetic causation in that biological siblings have a higher level of blood pressure concordance than adoptive siblings raised within the same family (Biron et al. (1976) Can. Med. Assoc. J. 114:773-4). Additionally, identical twin studies have demonstrated a higher concordance in blood pressure than that seen in fraternal twins (Christian, J. C. (1985) Ross Laboratories, Columbus, Ohio, pp. 51-55). However, in spite of these observations a number of epigenetic factors have also been reasoned to influence development of hypertension, including age, body mass, gender, and diet (Lifton, R. P, 1995).
Investigations into the etiology and inception of human hypertension have been centered around the use of inbred animal models of genetic hypertension, which present efficient, easily manipulatable systems for molecular and genetic analyses. Rodent models of hypertension include the spontaneously hypertensive rat (SHR), the stroke-prone SHR (SP-SHR), the Dahl salt-sensitive rat, the John Rapp salt-sensitive strain of rat, and numerous mouse strains (Dzau et al. (1995) Circulation 92(2):521-31). Advantages of using rodent models of hypertension include the genetic homogeneity achieved by fully inbred strains and the ability to produce cross-bred hybrid strains of predetermined genetic composition in suitably large populations (Hubner et al. (1995) Herz. 20:309-14).
The widely-used SHR has been studied in great detail. This animal model is characterized by a number of phenotypic abnormalities, including vascular and cardiac hypertrophy, and alterations in angiotensin responsiveness, which have been linked to the development and maintenance of hypertension (Yamori, Y. (1982) Hypertension. pp-556-81). Changes in the SHR cerebral microcirculation have also been reported (Herman, I. M. et al, (1988). Tissue and Cell. 20(1):1-12. The SHR is amenable for mapping of genes linked to hypertension due to its genetic homogeneity. To date, candidate loci include angiotensin-converting enzyme (Jacob et al. (1991) Cell. 67:213-24), neuropeptide Y (NYP) (Katsuya et al (1993) Biochem. Biophys. Res. Commun. 192:261-7), renin (Rapp et al (1989) Science. 243:542-4), guanylyl cyclase A/atrial natriuretic peptide receptor (GCA) (Krieger et al (1994) Hypertension 12:(S3):S66), heat shock protein 70 (hsp70) (Hamet et al (1992) Hypertension 19:611-4); and SA (Krieger et al. (1992) Hypertension 20:412). The results of these studies confirm that like essential hypertension in humans, hypertension in rodents is a polygenic disease. This reinforces the importance of animal modeling in trying to understand human disease to determine the molecular mechanism(s) by which the onset of hypertension occurs and how the process is maintained.
The present invention is based, at least in part, on the discovery of novel molecules which are differentially expressed in hypertensive humans, rats, and mice, referred to herein as xe2x80x9chypertension associated transcription factor-1xe2x80x9d (xe2x80x9cHATF-1xe2x80x9d) nucleic acid and protein molecules, as well as homologues thereof, referred to herein as xe2x80x9cHATF-1 Related Protein-1xe2x80x9d (xe2x80x9cHRP-1xe2x80x9d) nucleic acid and protein molecules. The HATF-1 and HRP-1 molecules of the present invention are useful as agents for diagnosing or prognosing subjects at risk for developing a cardiovascular disorder, e.g., hypertension, as well as modulating agents in regulating a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding HATF-1 and HRP-1 proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of HATF-1-encoding and HRP-1-encoding nucleic acids.
In one embodiment, an HATF-1 and HRP-1 nucleic acid molecule of the invention is at least 50%, 55%, 60%, 65%, 70%, 73%, 75%, 80%, 85%, 86%, 87%, 89%, 90%, 95%, 98%, or more homologous to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:1, 3, or 5 or a complement thereof.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:1, 3, or 5 or a complement thereof. In another preferred embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:1, 3, or 5. In another preferred embodiment, the nucleic acid molecule includes a fragment of at least 100 nucleotides of the nucleotide sequence of SEQ ID NO:1, 3, or 5 or a complement thereof.
Another embodiment of the invention features nucleic acid molecules, preferably HATF-1 and HRP-1 nucleic acid molecules, which specifically detect HATF-1 and HRP-1 nucleic acid molecules relative to nucleic acid molecules encoding non-HATF-1 and non-HRP-1 proteins, respectively. For example, in one embodiment, such a nucleic acid molecule is at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500 or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1, 3, or 5 or a complement thereof. In preferred embodiments, the nucleic acid molecules are at least 15 (e.g., contiguous) nucleotides in length and hybridize under stringent conditions to the nucleotide sequence of SEQ ID NO:1, 3, or 5 or a complement thereof.
Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to an HATF-1 or HRP-1 nucleic acid molecule, e.g., the coding strand of an HATF-1 or HRP-1 nucleic acid molecule.
Another aspect of the invention provides a vector comprising an HATF-1 or HRP-1 nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention.
In another aspect, the present invention provides a method for detecting the presence of an HATF-1 or HRP-1 nucleic acid molecule, protein or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting an HATF-1 or HRP-1 nucleic acid molecule, protein or polypeptide such that the presence of an HATF-1 or HRP-1 nucleic acid molecule, protein or polypeptide is detected in the biological sample.
In another aspect, the present invention provides a method for detecting the presence of HATF-1 or HRP-1 activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of HATF-1 or HRP-1 activity such that the presence of HATF-1 or HRP-1 activity is detected in the biological sample.
In another aspect, the invention provides a method for modulating HATF-1 or HRP-1 activity comprising contacting a cell capable of expressing HATF-1 or HRP-1 with an agent that modulates HATF-1 or HRP-1 activity such that HATF-1 or HRP-1 activity in the cell is modulated. In one embodiment, the agent inhibits HATF-1 or HRP-1 activity. In another embodiment, the agent stimulates HATF-1 or HRP-1 activity. In one embodiment, the agent is an antibody that specifically binds to an HATF-1 or HRP-1 protein. In another embodiment, the agent modulates expression of HATF-1 or HRP-1 by modulating transcription of an HATF-1 or HRP-1 gene or translation of an HATF-1 or HRP-1 mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of an HATF-1 or HRP-1 mRNA or an HATF-1 or HRP-1 gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant HATF-1 or HRP-1 protein or nucleic acid expression or activity by administering an agent which is an HATF-1 or HRP-1 modulator to the subject. In one embodiment the HATF-1 and HRP-1 modulator is an HATF-1 or HRP-1 nucleic acid molecule. In another embodiment, the HATF-1 or HRP-1 modulator is an HATF-1 or HRP-1 protein. In yet another embodiment, the HATF-1 or HRP-1 modulator is a peptide, peptidomimetic, or other small molecule. In a preferred embodiment, the disorder characterized by aberrant HATF-1 or HRP-1 protein or nucleic acid expression is a cardiovascular disorder, e.g., hypertension.
The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding an HATF-1 or HRP-1 protein; (ii) mis-regulation of the gene; and (iii) aberrant post-translational modification of an HATF-1 or HRP-1 protein, wherein a wild-type form of the gene encodes an protein with an HATF-1 or HRP-1 activity.
In another embodiment, the invention features an isolated protein, preferably an HATF-1 or HRP-1 protein, which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 73%, 75%, 80%, 85%, 86%, 87%, 89%, 90%, 95%, 98% or more homologous to a nucleotide sequence of SEQ ID NO:1, 3, 5 or a complement thereof. This invention further features an isolated protein, preferably an HATF-1 or HRP-1 protein, which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 5, or a complement thereof.
The proteins of the present invention or biologically active portions thereof, can be operatively linked to a non-HATF-1 or a non-HRP-1 polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins. The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferably HATF-1 and HRP-1 proteins. In addition, the HATF-1 and HRP-1 proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
In another embodiment, an HATF-1 and HRP-1 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2 or 4. In a preferred embodiment, an HATF-1 and HRP-1 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% 98% or more homologous to the amino acid sequence of SEQ ID NO:2 or 4.
In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of human, rat, or mouse HATF-1 or HRP-1. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:2 or 4.
In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or 4, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO: 1, 3, and/or 5 under stringent conditions.
Other features and advantages of the invention will be apparent from the following detailed description and claims.